Vaccine for preventing salmonella infection and application thereof

Abstract

The invention relates to a fusion protein for preventing salmonella infection, an immunogenic composition, a recombinant vaccine, a molecular architecture design, application and the like. According to the invention, three fusion proteins, namely PstS-LpfB, PstS-YidR and LpfB-SinH, which can be smoothly expressed in eukaryotic cells are selected to carry out animal immune experiments, so that the protection effect is verified. The invention finds that fusion protein molecules such as PstS-LpfB and the like can weaken tissue lesions caused by salmonella infection, have good immunogenicity, play a role in effective prevention and immune protection, efficiently prevent salmonella infection and have wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, particularly the field of immunopharmaceuticals, and specifically relates to a humoral immune vaccine for preventing Salmonella infection and its applications, etc. Background Art

[0002] Salmonella ( Salmonella ) is a Gram-negative bacillus widely distributed in nature and also present in the intestines of healthy humans and animals. When the body's immunity declines or long-term use of antibiotics leads to dysbacteriosis, infections can occur. The types of infections mainly include food poisoning, enteritis, typhoid fever, paratyphoid fever, septicemia, etc. In recent years, Salmonella has become an important foodborne pathogen and a potential pathogen that cannot be ignored in causing community-acquired infections and nosocomial infections in hospitals.

[0003] Salmonella is classified according to the antigen components on its cell surface, mainly including O antigen (lipopolysaccharide antigen) and H antigen (flagellar antigen). Currently, more than 2,600 serotypes of Salmonella have been discovered. Among them, Salmonella enteritidis ( Salmonella enteritidis ) and Salmonella typhimurium ( Salmonella typhimurium ) are the two most common serotypes related to human diseases. The proportion of these two serotypes in global human salmonellosis exceeds 40%, and in China, it exceeds 30%.

[0004] The World Health Organization (WHO) has listed Salmonella as one of the important threats to global public health. According to WHO reports, Salmonella causes more than 93.8 million cases of gastroenteritis and more than 155,000 deaths globally every year. WHO also emphasizes the issue of Salmonella drug resistance, pointing out that its drug resistance is continuously increasing and has become a serious public health problem. Therefore, developing effective Salmonella vaccines is of great significance for controlling its transmission and infection.

[0005] Due to the lack of commercialized vaccines, the current clinical treatment of Salmonella infections still mainly relies on antibiotic therapy. However, antibiotic treatment has precisely become the main driving force for the emergence and spread of highly drug-resistant and highly virulent strains. To break the current dilemma, researchers are actively developing new vaccines. For example, the prior art patent CN104736171B (publication date: November 6, 2018) discloses an inactivated Salmonella vaccine. By inactivating Salmonella enterica serotypes C1 or C2-3, a trivalent or tetravalent vaccine is prepared, providing a certain cross-immune protection effect; patent CN119241728A (publication date: November 14, 2024) discloses a method for preparing a fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen, but does not provide relevant data on vaccine efficacy testing. There are too many serotypes of pathogenic Salmonella. Prevention and control with traditional inactivated and attenuated vaccines have limited protective efficacy and are difficult to iterate. Although some studies have proposed candidate targets for subunit vaccine development, most have not verified the efficacy of candidate targets and subunit vaccines through animal experiments. Therefore, the development of Salmonella vaccines is still in a relatively primary stage and cannot meet the huge current market demand.

[0006] The phosphate-binding protein PstS (Phosphate-binding protein PstS) is part of the phosphate-specific transport (Pst) system, located in the bacterial periplasmic space, and has a high affinity for phosphate. Existing studies have proven that PstS can be used as a biosensor for phosphate pollution. For example, patent CN114965403A (publication date: August 30, 2022) discloses a method for constructing a phosphate biosensor using Pseudomonas aeruginosa PstS protein. In addition, in some pathogenic bacteria, the expression of PstS is related to the production of virulence factors and is involved in biofilm formation and host cell adhesion. However, there has been no study reporting that PstS can be used as a protective antigen for Salmonella and applied to vaccine development.

[0007] The molecular chaperone LpfB is a chaperone protein whose main function is to assist in the synthesis of long polar fimbriae (Lpf). Lpf is a fimbrial structure on the surface of Salmonella and plays an important role in the adhesion and colonization of Salmonella. Some studies have demonstrated that the LpfB protein can assist in the assembly and transportation of Lpf, enabling Salmonella to better adhere to host cells, especially M cells (microfold cells) in the Peyer's patch region of the small intestine, thereby promoting the colonization and infection of Salmonella in the host. Although some studies have attempted to use LpfB alone as a vaccine antigen, it was found that LpfB alone has no immunoprotective effect. However, there is no study demonstrating whether it can assist in enhancing the immunoprotective efficacy of the vaccine when this protein is fused with other antigens.

[0008] In summary, developing a vaccine that can provide broad-spectrum protection against Salmonella, is safe and effective, and is easy to produce has important scientific significance and application value. Such a vaccine can not only prevent various infections in humans but may also be applied to the prevention of animal diseases, aiming to reduce the incidence of infections caused by Salmonella, bringing significant benefits to public health and the economic level. Therefore, in view of the above problems, the present invention proposes a novel fusion protein design scheme aiming to develop an efficient and broad-spectrum vaccine for the prevention of Salmonella infection, which is expected to simultaneously address Salmonella infections of multiple different serotypes. Summary of the Invention

[0009] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a new fusion protein for the prevention of Salmonella infection, an immunogenic composition, a recombinant vaccine, and a molecular architecture design and application, etc. Starting from the perspective of reverse vaccinology, the present invention screens highly conserved target-binding immunogenic epitopes of Salmonella to determine candidate antigens for the design of fusion proteins. After in vitro expression verification, three fusion protein designs, namely PstS-LpfB, PstS-YidR, and LpfB-SinH, which can be successfully expressed in eukaryotic cells, are selected for animal immunization experiments to verify the protective effect. The present invention discovers that fusion protein molecules such as PstS-LpfB can attenuate tissue lesions caused by Salmonella infection, have good immunogenicity, play an effective role in prevention and immunoprotection, and can efficiently prevent Salmonella infection. The present invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, uses, etc.

[0010] One aspect of the present invention provides a fusion protein, characterized in that it is a fusion protein selected from any one of the following (1)-(3): (1)Fusion protein A; the fusion protein A contains a phosphate-binding protein PstS antigen or a fragment thereof; preferably, the fusion protein further contains a molecular chaperone LpfB antigen or a fragment thereof; (2)Fusion protein B: the fusion protein B contains the PstS antigen or a fragment thereof and a YidR antigen or a fragment thereof; (3)Fusion protein C: the fusion protein C contains the LpfB antigen or a fragment thereof and a SinH antigen or a fragment thereof.

[0011] Furthermore, the PstS, LpfB, YidR, SinH antigens or fragments thereof are derived from bacteria.

[0012] Furthermore, the bacteria are Gram-negative bacteria.

[0013] Furthermore, the Gram-negative bacteria are Enterobacteriaceae bacteria.

[0014] Furthermore, the Enterobacteriaceae bacteria are Salmonella.

[0015] Furthermore, the fusion protein A contains the PstS antigen or a fragment thereof and the LpfB antigen or a fragment thereof in sequence from the N-terminus to the C-terminus; the fusion protein B contains the PstS antigen or a fragment thereof and the YidR antigen or a fragment thereof in sequence from the N-terminus to the C-terminus; the fusion protein C contains the LpfB antigen or a fragment thereof and the SinH antigen or a fragment thereof in sequence from the N-terminus to the C-terminus.

[0016] Furthermore, the amino acid sequence of the PstS antigen or a fragment thereof is as shown in SEQ ID NO: 1, the amino acid sequence of the LpfB antigen or a fragment thereof is as shown in SEQ ID NO: 2, the amino acid sequence of the YidR antigen or a fragment thereof is as shown in SEQ ID NO: 9, and the amino acid sequence of the SinH antigen or a fragment thereof is as shown in SEQ ID NO: 10.

[0017] Furthermore, the N-terminus of the fusion protein further contains a signal peptide and / or the Fc domain of the human immunoglobulin heavy chain constant region γ1 protein (IGHG1); alternatively, the C-terminus of the fusion protein further contains the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit.

[0018] Furthermore, the signal peptide is derived from human Azurocidin protein, and its amino acid sequence is as shown in SEQ ID NO: 3; the amino acid sequence of the Fc domain of IGHG1 is as shown in SEQ ID NO: 4; the amino acid sequence of STABILON is as shown in SEQ ID NO.5.

[0019] Furthermore, different elements are optionally connected by GGS linker sequences, spacer sequences, and / or GS linker sequences.

[0020] Furthermore, the amino acid sequence of the spacer sequence is as shown in SEQ ID NO: 6, the amino acid sequence of the GGS linker sequence is as shown in SEQ ID NO: 7, and the amino acid sequence of the GS linker sequence is as shown in SEQ ID NO: 8.

[0021] Furthermore, the fusion protein comprises any one of the following 5 fusion proteins: (1) Fusion protein A: sequentially includes from the N-terminus to the C-terminus: signal peptide, Fc domain, GGS linker, PstS antigen protein or its fragment, spacer sequence, LpfB antigen or its fragment, GS linker, STABILON; (2) Fusion protein B: sequentially includes from the N-terminus to the C-terminus: signal peptide, Fc domain, GGS linker, PstS antigen protein or its fragment, spacer sequence, YidR antigen protein or its fragment, GS linker, STABILON; (3) Fusion protein C: sequentially includes from the N-terminus to the C-terminus: signal peptide, Fc domain, GGS linker, LpfB antigen protein or its fragment, spacer sequence, SinH antigen protein or its fragment, GS linker, STABILON; (4) Fusion protein D: sequentially includes from the N-terminus to the C-terminus: signal peptide, Fc domain, GGS linker, PstS antigen protein or its fragment, spacer sequence, SthB antigen protein or its fragment, GS linker, STABILON; (5) Fusion protein E: sequentially includes from the N-terminus to the C-terminus: signal peptide, Fc domain, GGS linker, SinH antigen protein or its fragment, spacer sequence, SthB antigen protein or its fragment, GS linker, STABILON; Furthermore, the amino acid sequence of the PstS antigen or its fragment is as shown in SEQ ID NO: 1, the amino acid sequence of the LpfB antigen or its fragment is as shown in SEQ ID NO: 2, the amino acid sequence of the YidR antigen or its fragment is as shown in SEQ ID NO: 9, the amino acid sequence of the SinH antigen or its fragment is as shown in SEQ ID NO: 10, the amino acid sequence of the SthB antigen or its fragment is as shown in SEQ ID NO: 11, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 3, and the amino acid sequence of the Fc domain is as shown in SEQ ID NO: 4; the amino acid sequence of the STABILON is as shown in SEQ ID NO. 5, the amino acid sequence of the spacer sequence is as shown in SEQ ID NO: 6, the amino acid sequence of the GGS linker sequence is as shown in SEQ ID NO: 7, and the amino acid sequence of the GS linker sequence is as shown in SEQ ID NO: 8.

[0022] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it comprises a nucleic acid encoding the fusion protein according to any one of the present invention.

[0023] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule according to the present invention.

[0024] Furthermore, the recombinant gene expression cassette further comprises one or more of a promoter, a terminator, and a regulatory sequence.

[0025] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule according to the present invention or the recombinant gene expression cassette according to the present invention.

[0026] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0027] Furthermore, the prokaryotic vector includes but is not limited to an Escherichia coli vector.

[0028] Furthermore, the Escherichia coli vector includes but is not limited to pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, pBR vector.

[0029] Furthermore, the eukaryotic vector includes but is not limited to a yeast expression vector, an insect expression vector, and a mammalian cell expression vector.

[0030] Furthermore, the yeast expression vector includes but is not limited to pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, pPIC9 vector.

[0031] Another aspect of the present invention provides a recombinant host cell, characterized by comprising the recombinant nucleic acid molecule of the present invention, or the recombinant gene expression cassette of the present invention, or the recombinant vector of the present invention.

[0032] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0033] Furthermore, the eukaryotic cell comprises a mammalian cell, an insect cell, or a yeast cell.

[0034] Furthermore, the yeast cell includes but is not limited to Saccharomyces cerevisiae, Pichia pastoris, and Hansenula yeast.

[0035] Furthermore, the prokaryotic cell includes but is not limited to Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0036] Furthermore, the Escherichia coli cells include but are not limited to BL21(DE3), DH5α, TOP10, and Rosetta.

[0037] Another aspect of the present invention provides an immunogenic composition or a pharmaceutical composition, characterized by comprising one or more of the fusion proteins of any one of the present invention, and / or one or more of the recombinant nucleic acid molecules of the present invention, and / or one or more of the recombinant gene expression cassettes of the present invention, and / or one or more of the recombinant vectors of the present invention, and / or one or more of the recombinant host cells of the present invention.

[0038] Furthermore, the immunogenic composition or the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0039] Furthermore, the immunogenic composition or the pharmaceutical composition further comprises other drugs for preventing diseases caused by Salmonella.

[0040] Furthermore, the other drugs for preventing diseases caused by Salmonella comprise antibiotics.

[0041] Another aspect of the present invention provides a recombinant vaccine, characterized by comprising one or more of the fusion proteins of any one of the present invention, and / or one or more of the recombinant nucleic acid molecules of the present invention, and / or one or more of the recombinant gene expression cassettes of the present invention, and / or one or more of the recombinant vectors of the present invention, and / or one or more of the recombinant host cells of the present invention, and / or one or more of the immunogenic compositions or pharmaceutical compositions of the present invention Furthermore, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine.

[0042] Furthermore, the recombinant vaccine is a nucleic acid vaccine.

[0043] Another aspect of the present invention provides the use of one or more fusion proteins of any one of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions of the present invention, and / or one or more recombinant vaccines of the present invention in the preparation of a medicament for preventing diseases caused by Salmonella.

[0044] Another aspect of the present invention provides a method for preventing diseases caused by Salmonella, which is characterized by comprising administering to a subject one or more fusion proteins of any one of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions of the present invention, and / or one or more recombinant vaccines of the present invention.

[0045] Furthermore, the diseases caused by Salmonella include but are not limited to gastroenteritis, typhoid fever, bacteremia, local suppurative infection, reactive arthritis, enteric fever, intravascular infection, focal infection.

[0046] Furthermore, the focal infection includes osteomyelitis, arthritis, meningitis, endocarditis, liver abscess, cholecystitis, pyelonephritis.

[0047] The fusion proteins, immunogenic compositions, recombinant vaccines, etc. of the present invention have the following beneficial technical effects: 1. The present invention discovers that the new fusion molecule has good immunogenicity and can provide an immune protection effect, and can be used for the research and development of nucleic acid vaccines or subunit vaccines. The immunogenic composition of the present invention can provide effective immune protection against Salmonella infection.

[0048] 2. The present invention can be used as a universal antigen framework for nucleic acid vaccines or subunit vaccines, has strong immunogenicity, and can induce broad-spectrum immune protection without being restricted by serotypes compared with traditional vaccines.

[0049] 3. Through systematic screening and combined with immunogenic epitope prediction analysis, the present invention finally identifies a total of 5 candidate antigens, namely PstS, LpfB, YidR, SthB, and SinH, for fusion protein design and experimental verification. And in order to improve the expression of candidate antigens and other characteristics such as enhancing the immunogenicity of antigens, N-terminal truncation or amino acid mutation at individual sites of the known antigens PstS, LpfB, YidR, SthB, and SinH is carried out.

[0050] 4. The present invention utilizes reverse vaccinology technology to screen candidate targets with broad-spectrum protective effects, preferably selects PstS antigen and LpfB antigen for vaccine design, and verifies through comparative experiments that the fusion protein composed of the two antigens can induce immune protection against Salmonella in mice.

[0051] 5. Example 5 of the present invention shows that the fusion molecule based on the present invention can effectively promote the high-abundance expression and secretion of bacterial proteins in eukaryotic cells, and the expressed proteins have correct and stable structures, which is beneficial for the presentation of immune epitopes, thereby further improving the immune protection effect and providing a new technology for the field of immunopharmaceutical design of multivalent Salmonella vaccines.

[0052] 6. The mouse gavage infection model experiment in Example 6 of the present invention shows that in the lung tissue, the bacterial load of the mouse group inoculated with the vaccine of the present invention is significantly reduced compared with the PBS control group. Statistical analysis shows that this reduction is significantly different (P<0.001). This result proves that the vaccine based on the present invention can effectively reduce the colonization of Salmonella in the intestine, spleen and liver, and provide effective multi-tissue immune protection.

[0053] 7. The fusion protein vaccine provided by the present invention exhibits excellent immunogenicity and protective effects, and has great potential in preventing Salmonella infection. This new vaccine design strategy provides a reference for the development of vaccines against other bacterial pathogens. Brief Description of the Drawings

[0055] Figure 1 It is a schematic diagram of the molecular structure of the vaccines used in the comparative experiment in the example, including the schematic diagrams of the molecular structures of Vaccine A, Vaccine B, Vaccine C, Vaccine D, and Vaccine E.

[0056] Figure 2 It is a schematic diagram of a circular plasmid containing the fusion protein molecular architecture of the present invention.

[0057] Figures 3A - 3E It is the quality control peak map and purity detection result of the nucleic acid vaccine used in the comparative experiment in the example of the present invention; among them, Figure 3A It is the quality control peak map and purity detection result of Vaccine A, Figure 3B It is the quality control peak map and purity detection result of Vaccine B, Figure 3C It is the quality control peak map and purity detection result of Vaccine C, Figure 3D It is the quality control peak map and purity detection result of Vaccine D, Figure 3E It is the quality control peak map and purity detection result of Vaccine E.

[0058] Figures 4A - 4DThese are the results of Western blot (WB) detection of the in vitro expression of vaccines A, B, C, D, and E of the present invention transfected into HEK293 cells. Among them, Figure 4A corresponds to the WB detection result of the in vitro expression of vaccine A transfected into HEK293 cells, Figure 4B corresponds to the WB detection result of the in vitro expression of vaccine B transfected into HEK293 cells, Figure 4C corresponds to the WB detection results of the in vitro expressions of vaccines C and D transfected into HEK293 cells, Figure 4D corresponds to the WB detection result of the in vitro expression of vaccine E transfected into HEK293 cells.

[0059] Figure 5 It is a schematic diagram of the immunization, challenge, and sampling processes of the mouse infection experiment.

[0060] Figures 6A - 6B These are the detection results of the bacterial loads in the feces of mice in each experimental group after immunization and challenge. Among them Figure 6A are the photos of the plates smeared with the bacterial loads in the feces of mice in each experimental group after immunization and challenge, Figure 6B is the analysis and comparison chart of the bacterial loads in the feces of mice in each experimental group after immunization and challenge.

[0061] Figures 7A - 7B These are the detection results of the bacterial loads in the spleen tissues of mice in each experimental group after immunization and challenge. Among them Figure 7A are the photos of the plates smeared with the bacterial loads in the spleen tissues of mice in each experimental group after immunization and challenge, Figure 7B is the analysis and comparison chart of the bacterial loads in the spleen tissues of mice in each experimental group after immunization and challenge.

[0062] Figures 8A - 8B These are the detection results of the bacterial loads in the liver tissues of mice in each experimental group after immunization and challenge. Among them Figure 8A are the photos of the plates smeared with the bacterial loads in the liver tissues of mice in each experimental group after immunization and challenge, Figure 8B is the analysis and comparison chart of the bacterial loads in the liver tissues of mice in each experimental group after immunization and challenge. Detailed implementation methods

[0063] Terms and definitions The term "Salmonella" refers to Salmonella, a common foodborne pathogenic bacterium belonging to the Enterobacteriaceae family, a Gram-negative bacillus with peritrichous flagella, capable of motility, and most having fimbriae. Salmonella is widely parasitic in the intestines of humans, poultry, and livestock and is mainly transmitted through contaminated food, such as raw poultry meat, eggs, beef, and certain fruits and vegetables.

[0064] The term "Salmonella infection" refers to various diseases caused by Salmonella, including but not limited to gastroenteritis, typhoid fever, bacteremia, local suppurative infections, reactive arthritis, etc.

[0065] The term "PstS" refers to the phosphate - specific transport system substrate - binding protein (abbreviated as PstS), which is an important component of the phosphate transport system in Salmonella. Preferably, the amino acid sequence of PstS is as shown in SEQ ID NO: 1.

[0066] The term "LpfB" refers to the molecular chaperone LpfB (abbreviated as LpfB), which is a chaperone protein whose main function is to assist in the synthesis of long polar fimbriae (Lpf). Lpf is a fimbrial structure on the surface of Salmonella and plays an important role in the adhesion and colonization of Salmonella. Preferably, the amino acid sequence of LpfB is as shown in SEQ ID NO: 2.

[0067] The term "YidR" refers to a conserved protein in Salmonella that contains a DUF3748 domain (DUF3748 - domain - containing protein). Its function has not been fully elucidated, but it may be involved in the stress response and biofilm formation of bacteria. Preferably, the amino acid sequence of YidR is as shown in SEQ ID NO: 9.

[0068] The term "SinH" refers to a protein in Salmonella that may be involved in regulating the virulence and adaptive responses of bacteria. This protein plays an important role in the interaction between bacteria and the host, and may promote the survival and pathogenicity of bacteria by regulating biofilm formation or affecting specific stress response pathways. Preferably, the amino acid sequence of SinH is as shown in SEQ ID NO: 10.

[0069] The term "SthB" refers to a protein in Salmonella that may be related to the homeostasis regulation and environmental adaptation of bacteria. Its specific function has not been fully clarified, and it may play a certain role in stress response, cell metabolism regulation, or pathogen adaptation. Preferably, the amino acid sequence of SthB is as shown in SEQ ID NO: 11.

[0070] The term "immune response" refers to the humoral response, cellular response, or both the humoral and cellular responses in an organism. The immune response can be measured by assays, including but not limited to assays for measuring the presence or amount of antibodies that specifically recognize proteins or cell - surface proteins, assays for measuring T - cell activation or proliferation, and / or assays for measuring the regulation of the activity or expression of one or more cytokines.

[0071] The terms "administration" or "inoculation" refer to administration preferably by the intramuscular or subcutaneous route of the nucleic acid vaccine or vaccine composition of the present invention, although other routes of administration can also be used, for example, oral, intranasal (e.g., aerosol or other non-injection administration), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or by combined routes.

[0072] The term "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0073] The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used for transformation into a suitable host cell.

[0074] The term "recombinant expression vector" refers to a DNA construct used for expressing, for example, a polynucleotide encoding a desired polypeptide. The recombinant expression vector can include, for example, a transcriptional subunit containing (1) a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structure or coding sequence that is transcribed into mRNA and translated into a protein; and (3) appropriate transcriptional and translational start and stop sequences.

[0075] The term "mRNA" refers to messenger RNA, Messenger RNA, the Chinese translation "messenger ribonucleic acid", which is a single-stranded ribonucleic acid transcribed from one strand of DNA as a template and carrying genetic information to direct protein synthesis.

[0076] The term "5'-UTR" refers to the "5' untranslated region" or "5'UTR", which is a part of a gene that is transcribed into a primary RNA transcript (precursor mRNA) and located upstream of the coding sequence.

[0077] The term "3'-UTR" refers to the "3'-untranslated region" or "3'UTR", which refers to the region located at the 3' end of a gene, downstream of the stop codon in the protein-coding region, and which is transcribed but not translated into an amino acid sequence, or refers to the corresponding region in an RNA molecule.

[0078] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such cells.

[0079] The terms "individual", "patient" or "subject" include mammals. Mammals include but are not limited to, domestic animals (e.g., pigs, cows, sheep, cats, dogs, and horses, etc.), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats, etc.).

[0080] The terms "transformation, transfection, transduction" have the meanings generally understood by those skilled in the art, that is, the process of introducing exogenous DNA or RNA into a host.

[0081] The term "drug combination" or "pharmaceutical composition" refers to auxiliary materials widely used in the field of drug production. The main purpose of using a carrier is to provide a drug composition that is safe to use, has stable properties and / or has specific functionality, and also to provide a method for effective absorption in the body of a subject.

[0082] The term "prevention" means that before suffering from a disease, by exposing (such as administering) a subject to a recombinant vaccine, composition, etc. based on the present invention, the symptoms after suffering from the disease are alleviated compared to when not exposed, and it does not necessarily mean complete inhibition of the disease.

[0083] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains.

[0084] The present invention discloses a method for preparing a new Salmonella vaccine and its application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention. The method and application of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0085] For the fusion protein, coding nucleic acid and its elements, preparation method and application provided by the present invention, the raw materials and reagents used can all be obtained commercially. Based on the basic knowledge of conventional molecular cloning, expression construction, vaccine preparation, immunology, etc. in the art, those skilled in the art can all implement the methods of the examples of the present invention.

[0086] The following further illustrates the present invention in conjunction with examples. Among them, as a preference, a nucleic acid vaccine framework is selected for preparing a recombinant vaccine.

[0087] Example 1 Antigen Screening Due to the limitations of serotypes of Salmonella vaccines, the present invention adopts a reverse vaccinology strategy to screen potential cross-immunoprotective antigens of Salmonella. The main goal of the screening is to find antigens that are highly expressed in the transcriptome and highly conserved among different Salmonella subtypes.

[0088] The specific screening steps are as follows: (1) Collect transcriptome data of Salmonella of different serotypes.

[0089] (2) Perform bioinformatics analysis on transcriptome data to identify genes with high abundance expression.

[0090] (3) Using comparative genomics methods, we analyzed the conservation of these highly expressed genes among different serotypes.

[0091] After the above-mentioned systematic screening, combined with immune epitope prediction analysis, the present invention finally identified PstS, LpfB, YidR, SthB, and SinH, a total of 5 antigens for subsequent fusion protein design and experimental verification.

[0092] In addition, in order to improve the expression of candidate antigens and enhance the immunogenicity of antigens, the known PstS, LpfB, YidR, SthB, and SinH antigens were N-terminally truncated or amino acid mutations were performed at individual sites.

[0093] Example 2 Fusion protein vaccine design and recombinant nucleic acid vaccine construction According to the screening results in Example 1, a total of five fusion protein vaccines were obtained through fusion protein design: PstS-LpfB, PstS-YidR, LpfB-SinH, PstS-SthB, and SinH-SthB.

[0094] In order to prepare the above five recombinant nucleic acid vaccines, the schematic diagram of the vaccine molecular architecture involved in the embodiment is shown as follows: Figure 1 As shown, the following elements are included from N-terminus to C-terminus: signal peptide, Fc domain, target antigen region, STABILON. Each element can be directly connected or connected through different linkers, such as GGS linker, spacer sequence or GS linker. Figure 1 The five vaccines shown in the figure, namely, vaccine A, vaccine B, vaccine C, vaccine D, and vaccine E, differ in that their target antigen regions are different. The target antigen of vaccine A is PstS-LpfB (the two can be connected via a spacer sequence), the target antigen of vaccine B is PstS-YidR (the two can be connected via a spacer sequence), the target antigen of vaccine C is LpfB-SinH (the two can be connected via a spacer sequence), the target antigen of vaccine D is PstS-SthB (the two can be connected via a spacer sequence), and the target antigen of vaccine E is SinH-SthB (the two can be connected via a spacer sequence). In order to prepare vaccines that can produce Figure 1The recombinant nucleic acid vaccine of the protein shown. First, a gene expression cassette is constructed for expressing the antigen sequence of the fusion protein molecular architecture described in the present invention. The expression cassette sequentially includes from the 5'-end to the 3'-end: 5'-UTR, CDS region, 3'-UTR, PolyA, wherein the CDS region contains the fusion protein molecular architecture described in the present invention. Subsequently, based on codon degeneracy, the complete gene expression cassette sequence is optimized, and the DNA sequence is directly obtained by gene synthesis (commissioned by GenScript Corporation for synthesis). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector available for in vitro RNA transcription, such as Figure 2 as shown, to obtain the vector plasmid for preparing the recombinant nucleic acid vaccine.

[0095] According to the above method, the vector for subsequent examples is prepared: (1) Vector for preparing recombinant nucleic acid vaccine A containing PstS-LpfB Step a: Synthesize the "signal peptide - Fc domain of human IGHG - PstS antigen - LpfB antigen - STABILON" fusion gene fragment. The Fc domain of human IGHG and the PstS antigen are connected by the GGS linker sequence shown in SEQ ID NO: 7, the PstS antigen - LpfB antigen are connected by the spacer sequence shown in SEQ ID NO: 6, the LpfB antigen and STABILON are connected by the GS linker sequence shown in SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3, the amino acid sequence of the Fc domain of human IGHG is shown in SEQ ID NO: 4, the amino acid sequence of the PstS antigen is shown in SEQ ID NO.1, the amino acid sequence of the LpfB antigen is shown in SEQ ID NO.2, and the amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0096] Step b: Construct the nucleic acid vaccine architecture vector.

[0097] The nucleic acid vaccine architecture vector contains 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0098] Step c: Prepare the recombinant plasmid.

[0099] Insert the gene synthesized in step a into the vector architecture of step b to obtain the vector for preparing recombinant nucleic acid vaccine A containing PstS-LpfB ( Figure 2 as shown).

[0100] (2) Vector for preparing recombinant nucleic acid vaccine B containing PstS-YidR Step a: Synthesize the "signal peptide - Fc domain of human IGHG - PstS antigen - YidR antigen - STABILON" fusion gene fragment. The Fc domain of human IGHG and the PstS antigen are linked by the GGS linker sequence shown in amino acid sequence SEQ ID NO: 7. The PstS antigen and the YidR antigen are linked by the spacer sequence shown in amino acid sequence SEQ ID NO: 6. The YidR antigen and STABILON are linked by the GS linker sequence shown in amino acid sequence SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3. The amino acid sequence of the Fc domain of human IGHG is shown in SEQ ID NO: 4. The amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1. The amino acid sequence of the YidR antigen is shown in SEQ ID NO: 9. The amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0101] Step b: Construct a nucleic acid vaccine framework vector.

[0102] The nucleic acid vaccine framework vector contains 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0103] Step c: Prepare a recombinant plasmid.

[0104] Insert the gene synthesized in step a into the vector framework of step b to obtain a recombinant nucleic acid vaccine B preparation vector containing PstS - YidR ( Figure 2 as shown).

[0105] (3) Recombinant nucleic acid vaccine C preparation vector containing LpfB - SinH Step a: Synthesize the fusion gene fragment of "signal peptide - Fc domain of human IGHG - LpfB antigen - SinH antigen - STABILON". The Fc domain of human IGHG and the LpfB antigen are connected by the GGS linker sequence shown in the amino acid sequence SEQ ID NO: 7. The LpfB antigen and the SinH antigen are connected by the spacer sequence shown in the amino acid sequence SEQ ID NO: 6. The SinH antigen and STABILON are connected by the GS linker sequence shown in the amino acid sequence SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3. The amino acid sequence of the Fc domain of human IGHG is shown in SEQ ID NO: 4. The amino acid sequence of the LpfB antigen is shown in SEQ ID NO: 2. The amino acid sequence of the SinH antigen is shown in SEQ ID NO: 10. The amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0106] Step b: Construct a nucleic acid vaccine framework vector.

[0107] The nucleic acid vaccine framework vector contains 5'-UTR and 3'-UTR and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0108] Step c: Prepare a recombinant plasmid.

[0109] Insert the gene synthesized in step a into the vector framework of step b to obtain a recombinant nucleic acid vaccine C preparation vector containing LpfB - SinH ( Figure 2 as shown).

[0110] (4) Recombinant nucleic acid vaccine D preparation vector containing PstS - SthB Step a: Synthesize the "signal peptide - Fc domain of human IGHG - PstS antigen - SthB antigen - STABILON" fusion gene fragment. The Fc domain of human IGHG and the PstS antigen are linked by the GGS linker sequence shown in amino acid sequence SEQ ID NO: 7. The PstS antigen and the SthB antigen are linked by the spacer sequence shown in amino acid sequence SEQ ID NO: 6. The SthB antigen and STABILON are linked by the GS linker sequence shown in amino acid sequence SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3. The amino acid sequence of the Fc domain of human IGHG is shown in SEQ ID NO: 4. The amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1. The amino acid sequence of the SthB antigen is shown in SEQ ID NO: 11. The amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0111] Step b: Construct a nucleic acid vaccine framework vector.

[0112] The nucleic acid vaccine framework vector contains 5'-UTR and 3'-UTR and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0113] Step c: Prepare a recombinant plasmid.

[0114] Insert the gene synthesized in step a into the vector framework of step b to obtain a recombinant nucleic acid vaccine D preparation vector containing PstS - SthB ( Figure 2 as shown).

[0115] (5) Recombinant nucleic acid vaccine E preparation vector containing SinH - SthB Step a: Synthesize the fusion gene fragment of "signal peptide - Fc domain of human IGHG - SinH antigen - SthB antigen - STABILON". The Fc domain of human IGHG and the SinH antigen are linked by the GGS linker sequence shown in SEQ ID NO: 7 in terms of amino acid sequence. The SinH antigen and the SthB antigen are linked by the spacer sequence shown in SEQ ID NO: 6 in terms of amino acid sequence. The SthB antigen and STABILON are linked by the GS linker sequence shown in SEQ ID NO: 8 in terms of amino acid sequence. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3, the amino acid sequence of the Fc domain of human IGHG is shown in SEQ ID NO: 4, the amino acid sequence of the SinH antigen is shown in SEQ ID NO: 10, the amino acid sequence of the SthB antigen is shown in SEQ ID NO: 11, and the amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0116] Step b: Construct a nucleic acid vaccine framework vector.

[0117] The nucleic acid vaccine framework vector contains 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0118] Step c: Prepare a recombinant plasmid.

[0119] Insert the gene synthesized in step a into the vector framework of step b to obtain a recombinant nucleic acid vaccine E preparation vector containing SinH - SthB ( Figure 2 as shown).

[0120] Table 1 Protein amino acid sequences involved in the present invention

[0121] Example 3 Preparation of the recombinant nucleic acid vaccine of the present invention (1) Preparation of capped mRNA vaccine Step a: Digest the vector plasmid used for producing the capped mRNA vaccine in Example 2 to linearize it, and obtain a linearized plasmid for in vitro transcription.

[0122] Step b: Perform an in vitro co-transcription capping reaction on the linearized plasmid, add the 7-methylguanylate cap structure to the 5' end of the transcribed mRNA, and degrade the template DNA.

[0123] (2) Preparation of uncapped mRNA vaccine Step a: Digest the vector plasmid used for producing the uncapped mRNA vaccine in Example 2 to linearize it, and obtain a linearized plasmid for in vitro transcription.

[0124] Step b: Perform an in vitro uncapped transcription reaction on the linearized plasmid and degrade the template DNA.

[0125] (3) Preparation of DNA vaccine Step a: Amplify the vector plasmid used for producing the DNA vaccine in Example 2 to obtain a large amount of target plasmid for purification.

[0126] Step b: Extract and purify the target plasmid using an endotoxin-removing plasmid extraction and purification kit.

[0127] Example 4 Quality control of in vitro transcription of recombinant nucleic acid of the present invention and vaccine preparation Vaccine A (recombinant nucleic acid vaccine A containing PstS-LpfB), Vaccine B (recombinant nucleic acid vaccine B containing PstS-YidR), Vaccine C (recombinant nucleic acid vaccine C containing LpfB-SinH), Vaccine D (recombinant nucleic acid vaccine D containing PstS-SthB), and Vaccine E (recombinant nucleic acid vaccine E containing SinH-SthB) were prepared by the method for preparing the capped mRNA vaccine in Example 3. The purity of the produced recombinant nucleic acid was detected, and the purity of the recombinant nucleic acid used in the experiment was greater than or equal to 85%. The quality control peak map of the recombinant nucleic acid based on the present invention is as Figures 3A - 3E shown. Specifically described as: (1) Recombinant nucleic acid vaccine A containing PstS-LpfB, with a purity of 90.1%, and the quality control peak map and purity detection results are as Figure 3A ; (2) Recombinant nucleic acid vaccine B containing PstS-YidR, with a purity of 85.1%, and the quality control peak map and purity detection results are as Figure 3B ; (3) Recombinant nucleic acid vaccine C containing LpfB-SinH, with a purity of 89.3%, and the quality control peak map and purity detection results are as Figure 3C ; (4) Recombinant nucleic acid vaccine D containing PstS-SthB, with a purity of 85.4%, and the quality control peak map and purity detection results are as Figure 3D ; (5) Recombinant nucleic acid vaccine E containing SinH-SthB, with a purity of 90.7%, and the quality control peak map and purity detection results are as Figure 3E . The above purities all meet the quality requirements for cell transfection experiments and vaccine production.

[0128] Example 5 In vitro expression effect of the recombinant nucleic acid of the present invention The vaccines in Example 4 were transfected into HEK293T cells through a cell transfection reagent. After in vitro culture for 48 hours, the proteins were collected, subjected to Western blot detection, and the molecular weights of the proteins of Vaccine A, Vaccine B, Vaccine C, Vaccine D, and Vaccine E were calculated, as shown in Table 2.

[0129] Table 2 Protein Molecular Weights of Vaccine A, Vaccine B, Vaccine C, and Vaccine D Vaccine Protein molecular weight (kDa) A 89.3 B 111.8 C 84.6 D 97 E 92.3 Figures 4A - 4D The in vitro expression WB (Western blot) detection results of vaccines A, B, C, D, and E transfected into HEK293 cells are shown (where, Figure 4A corresponds to the in vitro expression WB detection result of vaccine A transfected into HEK293 cells, Figure 4B corresponds to the in vitro expression WB detection result of vaccine B transfected into HEK293 cells, Figure 4C corresponds to the in vitro expression WB detection results of vaccines C and D transfected into HEK293 cells, Figure 4D corresponds to the in vitro expression WB detection result of vaccine E transfected into HEK293 cells). The expressed antigens are all humoral immune antigens and theoretically significant expression can be detected in the supernatant.

[0130] As shown in Figure 4, the target protein signals of vaccines A, B, and C were detected in the supernatant, and the molecular weight sizes were all in line with expectations. This proves that the fusion protein based on the present invention can not only be successfully translated and correctly folded in eukaryotic cells, but also has a stable structure and can be secreted extracellularly. The target protein signals of vaccines D and E were not detected in the supernatant, proving that the designs of the two fusion proteins PstS-SthB and SinH-SthB cannot be normally secreted and expressed.

[0131] Therefore, excluding vaccines D and E that cannot be normally expressed and secreted, vaccines A, B, and C were selected for subsequent animal experiments to verify the immune protection effect.

[0132] Example 6 Preventive Effect of the Recombinant Nucleic Acid Vaccine Based on the Present Invention in a Mouse Gavage Infection Model To verify whether the nucleic acid vaccine based on the present invention has an immune protection effect, in this example, mice immunized with vaccine A (vaccine A immunization group), mice immunized with vaccine B (vaccine B immunization group), mice immunized with vaccine C (vaccine C immunization group) were respectively compared with non-immunized mice (PBS group) in an immunization and challenge experiment. One mouse was not immunized and not challenged as a negative control group.

[0133] Twenty-one 6-8-week-old BALB / C mice weighing 18-25 g were selected for the experiment. They were all housed in independent cages with constant temperature and humidity, and were allowed to adapt to the environment for 7 days in advance. The temperature in the breeding room was 20-26 °C, the humidity was 40-70%, with alternating light and darkness day and night. There was light from 8 am to 8 pm and darkness from 8 pm to 8 am the next day. Sufficient feed was continuously provided, and the mice could freely ingest it without limit. They drank sterile water, and the water bottles were continuously supplied with water for free ingestion. After the mice were adaptively bred, they were randomly grouped, and each mouse was marked with an ear tag. Specifically, as shown in Table 3, the dose in this table and in the following text refers to the amount of the active ingredient.

[0134] Table 3 Grouping and Immunization Process of Mouse Immunization Experiment in Example 5

[0135] The mice in each group were immunized twice according to the immunization process in Table 3. At Day34, the mice were gavaged with 100 μL (200 mg / mL) of streptomycin solution to enhance the effect of bacterial challenge. At Day35, the challenge was carried out. The mice were fasted 4 h before bacterial challenge. The mice were gavaged with 100 μL of Salmonella bacterial solution (1×10 7 CFU), and after gavaging, they were normally bred. The clinical manifestations of the mice were observed, monitored and recorded every day. At Day40, the mice were sacrificed for sampling, and the bacterial load in different tissues was detected to evaluate the immune protection effect of the vaccine. The immunization, challenge and sampling schedule is as Figure 5 shown.

[0136] Equal amounts of feces, spleen and liver tissues of each mouse were taken, added with sterile PBS buffer, and ground on ice until there was no obvious precipitate. The blood was diluted 1:1000 with sterile PBS buffer, and after mixing, 100 μl of the diluted solution was evenly spread on the solid medium and cultured for 24 h. The culture dish was taken out to count and photograph the bacterial clones, and the protective effect of the vaccine on the mice was observed. The results are shown in Figures 6, 7 and 8.

[0137] Figure 6 shows the comparison of the bacterial load in the feces of each experimental group after mouse immunization and challenge. Among them, Figure 6A is the plate photo of the bacterial load in the feces of the mice in each experimental group after immunization and challenge, Figure 6B is the analysis and comparison chart of the bacterial load in the feces of the mice in each experimental group after immunization and challenge. The results show that the bacterial load in the negative control group is 0, and the comparative experiment is valid. Compared with the mice in the PBS group, the Salmonella load in the mice immunized with Vaccine A decreased significantly, about 3 times; the bacterial load in the mice immunized with Vaccine B and Vaccine C did not decrease.

[0138] Figure 7 shows the comparison of the bacterial load in the spleen tissues of each experimental group after mouse immunization and challenge. Among them, Figure 7A is the plate photo of the bacterial load in the spleen tissues of the mice in each experimental group after immunization and challenge, Figure 7BIt is a comparative analysis chart of the bacterial loads in the spleen tissues of mice in each experimental group after immune challenge. The results show that the bacterial load in the negative control group is 0, indicating that the comparative experiment is valid. Compared with the mice in the PBS group, the Salmonella load in the mice immunized with Vaccine A decreased significantly, by about 7-fold; the bacterial load in the mice immunized with Vaccine B did not decrease; the bacterial load in the mice immunized with Vaccine C decreased.

[0139] Figure 8 shows the comparison of the bacterial loads in the livers of mice in each experimental group after immune challenge, where Figure 8A is a plate photo of the bacterial loads in the liver tissues of mice in each experimental group after immune challenge, Figure 8B is a comparative analysis chart of the bacterial loads in the liver tissues of mice in each experimental group after immune challenge. The results show that the bacterial load in the negative control group is 0, indicating that the comparative experiment is valid. Compared with the mice in the PBS group, the Salmonella load in the mice immunized with Vaccine A decreased significantly, by about 50-fold; the bacterial load in the mice immunized with Vaccine B did not decrease; the bacterial load in the mice immunized with Vaccine C decreased.

[0140] In summary, among the three fusion proteins PstS-LpfB, PstS-YidR, and LpfB-SinH provided by the present invention, PstS-LpfB is the best combination, which can induce effective protective immunity in the model animal mice and has a good preventive effect against Salmonella infection. The PstS-LpfB fusion protein is a preferred vaccine target. Therefore, the present invention provides a preferred antigen combination, which can be applied to vaccine research and development, filling the gap in the current field of Salmonella vaccine research and development, and having extremely high commercial value and broad application prospects.

[0141] The above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, rather than limiting the implementation manners of the present disclosure. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present disclosure shall be included in the protection scope of the claims of the present disclosure.

Claims

1. A fusion protein, characterized in that A fusion protein selected from any one of the following (1)-(3): Fusion protein A; the fusion protein A comprises a phosphate-binding protein PstS (Phosphate-binding protein PstS) antigen or a fragment thereof; preferably, the fusion protein further comprises a molecular chaperone LpfB (molecular chaperone LpfB) antigen or a fragment thereof; Fusion protein B: the fusion protein B comprises the PstS antigen or a fragment thereof and the YidR antigen or a fragment thereof; Fusion protein C: The fusion protein C comprises the LpfB antigen or a fragment thereof and the SinH antigen or a fragment thereof.

2. The fusion protein according to claim 1, characterized in that The PstS, LpfB, YidR, SinH antigens or fragments thereof are derived from bacteria; preferably, the bacteria are Gram-negative bacteria; preferably, the Gram-negative bacteria are Enterobacteriaceae; preferably, the Enterobacteriaceae are Salmonella.

3. The fusion protein according to claims 1 and 2, characterized in that The fusion protein A comprises the PstS antigen or a fragment thereof, and the LpfB antigen or a fragment thereof, in sequence from the N-terminus to the C-terminus; the fusion protein B comprises the PstS antigen or a fragment thereof, and the YidR antigen or a fragment thereof, in sequence from the N-terminus to the C-terminus; the fusion protein C comprises the LpfB antigen or a fragment thereof, and the SinH antigen or a fragment thereof, in sequence from the N-terminus to the C-terminus; preferably, the amino acid sequence of the PstS antigen or a fragment thereof is as shown in SEQ ID NO: 1, the amino acid sequence of the LpfB antigen or a fragment thereof is as shown in SEQ ID NO: 2, the amino acid sequence of the YidR antigen or a fragment thereof is as shown in SEQ ID NO: 9, and the amino acid sequence of the SinH antigen or a fragment thereof is as shown in SEQ ID NO:

10.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The N-terminus of the fusion protein further comprises a signal peptide and / or the Fc domain of human immunoglobulin heavy chain constant region γ1 protein (IGHG1); or, the C-terminus of the fusion protein further comprises the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit.

5. The fusion protein according to claim 4, characterized in that The signal peptide is derived from human Azurocidin protein, and its amino acid sequence is shown in SEQ ID NO: 3; the amino acid sequence of the Fc domain of IGHG1 is shown in SEQ ID NO: 4; and the amino acid sequence of STABILON is shown in SEQ ID NO.

5.

6. The fusion protein according to any one of claims 1 to 5, characterized in that Different elements are optionally connected through a GGS linker sequence, a spacer sequence, and / or a GS linker sequence; preferably, the amino acid sequence of the spacer sequence is as shown in SEQ ID NO: 6, the amino acid sequence of the GGS linker sequence is as shown in SEQ ID NO: 7, and the amino acid sequence of the GS linker sequence is as shown in SEQ ID NO:

8.

7. The fusion protein according to any one of claims 1 to 6, characterized in that Contains any one of the following five fusion proteins: (1) Fusion protein A: from N-terminus to C-terminus, it includes: signal peptide, Fc domain, GGS linker, PstS antigen protein or its fragment, spacer sequence, LpfB antigen or its fragment, GS linker, STABILON; (2) Fusion protein B: from N-terminus to C-terminus, it includes: signal peptide, Fc domain, GGS linker, PstS antigen protein or its fragment, spacer sequence, YidR antigen protein or its fragment, GS linker, STABILON; (3) Fusion protein C: from N-terminus to C-terminus, it includes: signal peptide, Fc domain, GGS linker, LpfB antigen protein or its fragment, spacer sequence, SinH antigen protein or its fragment, GS linker, STABILON; (4) Fusion protein D: from N-terminus to C-terminus, it includes: signal peptide, Fc domain, GGS linker, PstS antigen protein or its fragment, spacer sequence, SthB antigen protein or its fragment, GS linker, STABILON; (5) Fusion protein E: from N-terminus to C-terminus, it includes: signal peptide, Fc domain, GGS linker, SinH antigen protein or its fragment, spacer sequence, SthB antigen protein or its fragment, GS linker, STABILON; Preferably, the amino acid sequence of the PstS antigen or a fragment thereof is as shown in SEQ ID NO: 1, the amino acid sequence of the LpfB antigen or a fragment thereof is as shown in SEQ ID NO: 2, the amino acid sequence of the YidR antigen or a fragment thereof is as shown in SEQ ID NO: 9, the amino acid sequence of the SinH antigen or a fragment thereof is as shown in SEQ ID NO: 10, the amino acid sequence of the SthB antigen or a fragment thereof is as shown in SEQ ID NO: 11, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 3, and the amino acid sequence of the Fc domain is as shown in SEQ ID NO: 4; the amino acid sequence of the STABILON is as shown in SEQ ID NO.5, the amino acid sequence of the spacer sequence is as shown in SEQ ID NO: 6, the amino acid sequence of the GGS linker sequence is as shown in SEQ ID NO: 7, and the amino acid sequence of the GS linker sequence is as shown in SEQ ID NO:

8.

8. A recombinant nucleic acid molecule, characterized in that Comprising a nucleic acid encoding the fusion protein according to any one of claims 1 to 7.

9. A recombinant gene expression cassette, characterized in that: Comprising the recombinant nucleic acid molecule of claim 8.

10. A recombinant vector, characterized in that: Comprising the recombinant nucleic acid molecule of claim 8 or the recombinant gene expression cassette of claim 9.

11. A recombinant host cell, characterized in that Comprising the recombinant nucleic acid molecule of claim 8, or the recombinant gene expression cassette of claim 9, or the recombinant vector of claim 10.

12. An immunogenic composition or a pharmaceutical composition, characterized in that: Comprising one or more fusion proteins according to any one of claims 1 to 7, and / or one or more recombinant nucleic acid molecules according to claim 8, and / or one or more recombinant gene expression cassettes according to claim 9, and / or one or more recombinant vectors according to claim 10, and / or one or more recombinant host cells according to claim 11; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier; more preferably, the immunogenic composition or pharmaceutical composition further comprises other drugs for preventing diseases caused by Salmonella; most preferably, the other drugs for preventing diseases caused by Salmonella comprise antibiotics.

13. A recombinant vaccine, characterized in that: Comprising one or more fusion proteins of any one of claims 1 to 7, and / or one or more recombinant nucleic acid molecules of claim 8, and / or one or more recombinant gene expression cassettes of claim 9, and / or one or more recombinant vectors of claim 10, and / or one or more recombinant host cells of claim 11, and / or one or more immunogenic compositions or pharmaceutical compositions of claim 12; preferably, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine; more preferably, the recombinant vaccine is a nucleic acid vaccine.

14. Use of one or more fusion proteins according to any one of claims 1 to 7, and / or one or more recombinant nucleic acid molecules according to claim 8, and / or one or more recombinant gene expression cassettes according to claim 9, and / or one or more recombinant vectors according to claim 10, and / or one or more recombinant host cells according to claim 11, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 12, and / or one or more recombinant vaccines according to claim 13 in the preparation of a medicament for preventing a disease caused by Salmonella.

15. The use according to claim 14, characterized in that The diseases caused by the Salmonella include but are not limited to gastroenteritis, typhoid fever, bacteremia, local suppurative infection, reactive arthritis, enteric fever, intravascular infection, focal infection; preferably, the focal infection includes osteomyelitis, arthritis, meningitis, endocarditis, liver abscess, cholecystitis, pyelonephritis.

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